Genetic Inferences and Breeding Implications from Analysis of Cranberry Germplasm Anthocyanin Profi Les
Total Page：16
File Type：pdf, Size：1020Kb
J. AMER. SOC. HORT. SCI. 128(5):691–697. 2003. Genetic Inferences and Breeding Implications from Analysis of Cranberry Germplasm Anthocyanin Profi les Nicholi Vorsa1 and James Polashock2 Philip E. Marucci Center for Blueberry and Cranberry Research and Extension, Rutgers University, Chatsworth, NJ 08019 David Cunningham3 and Robin Roderick4 Ocean Spray Cranberries, Inc., One Ocean Spray Drive, Lakeville-Middleboro, MA 02349 ADDITIONAL INDEX WORDS. Vaccinium macrocarpon, fruit, methoxylation, glycosylation, cyanidin, peonidin ABSTRACT. A diversity of anthocyanins exists among angiosperm species. Studies indicate that various anthocyanins differ in antioxidant potential, their bioavailability, and stability during processing. The fruit of the American cranberry, Vaccinium macrocarpon Ait., is recognized as having six anthocyanins, composed largely of 3-O-galactosides and 3-O-arabinosides, and to lesser amount (≈6%), 3-O-glucosides of the aglycones cyanidin and peonidin. This study analyzed proportions of these six anthocyanins from >250 accessions of a germplasm collection over harvest dates. Fruit samples from 78 selected accessions, based on the fi rst year analysis, were also analyzed a second year. Principal component analysis identifi ed general negative relationships between the proportions of cyanidin versus peonidin, arabinosides versus glucosides, and galactosides versus arabinosides and glucosides. These relationships were consistent across the 2 years. Most variation in germplasm anthocyanin profi les refl ected variation of cyanidin versus peonidin proportions, with cyanidin to peonidin ratios ranging from 3.6:1 to 0.5:1. Variation for glycosylation profi les was also evident, with galactoside proportions ranging from 64% to 75%, arabinoside proportions ranging from 20% to 33%, and glucoside proportions ranging from 3% to 9%. Evidence for both signifi cant qualitative and quantitative genetic variation exists for the methoxylation of cyanidin to peonidin. Signifi cant quantitative genetic variation is also apparent for glycosylation. Signifi cant differences exist among anthocyanins as to their Wrolstad, 1990). Although total anthocyanin content (TAcy) of antioxidant potential (Cao et al., 1996). Antioxidant potential ap- fruit is one of the major traits evaluated in cranberry breeding pears to be a function of both the glycoside, as well as the specifi c (Sapers et al., 1983a), information on variation of proportions aglycone, i.e. cyanidin, malvidin, pelargonin, etc. (Satue-Gracia of individual anthocyanins is generally lacking. Earlier studies et al., 1997; Wang et al., 1997). For example, Wang et al. (1997) concluded that there were either no or very little qualitative or found cyanidin-3-glucoside to have ≈75% greater antioxidant quantitative differences in the proportions of individual antho- activity than cyanidin-3-galactoside. Cyanidin-3-glucoside was cyanins among the cranberry varieties or harvest dates studied found to have >90% greater antioxidant activity than peonidin- (Sapers et al.,1983b; Sapers and Hargrave, 1987). These studies 3-glucoside (Wang et al., 1997). Evidence is also increasing for did not consider the proportions of glucosides, most likely assum- the signifi cant role that the carbohydrate moiety plays in the ing they were of little consequence. The proportion of cyanidin to absorption of numerous fl avonoids in the gastro-intestinal tract peonidin anthocyanins was determined to be about equal (Hong (Gee et al., 2000; Hollman and Katan, 1997; Hollman et al., 1997; and Wrolstad, 1986, 1990; Sapers and Hargrave, 1987), however, Setchell et al., 2001). Glucoside conjugates appear to be the most these studies evaluated only a limited number of varieties. bioavailable. Hollman and Katan (1997) found that quercetin- We have assembled a large collection of cranberry germplasm 3-glucoside was absorbed to a signifi cantly greater degree than accessions from cultivated beds. Analyzing anthocyanin profi les quercetin-3-rutinoside in human subjects. Mizuma et al. (1994) of a broad array of germplasm over harvest dates and years may in studies with rat intestines found fl avonols as glucosides were provide insight into the genetic variation and inter-relationships absorbed to a greater degree than the galactosides. Anthocyanins among the specifi c anthocyanins in cranberry, as well as providing also differ in their stability during processing (Wrolstad et al., useful information for breeding of desirable anthocyanin profi les. 1994). The objectives of this study were to 1) analyze the proportions The American cranberry (Vaccinium macrocarpon Ait.) is of the individual anthocyanins in a broad array of germplasm recognized for its brilliant red fruit, which is a result of a fairly obtained from cultivated beds over harvest dates and years to high abundance of anthocyanins in the fruit epidermal tissues. The better assess the variation for anthocyanin profi les in American anthocyanins of V. macrocarpon are mainly the 3-O-glycosides cranberry, 2) identify relationships, or possible tradeoffs, among of cyanidin and peonidin. Galactosides and arabinosides of cy- the anthocyanins using principal component analysis, and 3) iden- anidin and peonidin have been reported to be the most abundant, tify varieties with non-typical anthocyanin profi les. with lesser (≈6%) amounts of glucoside anthocyanins (Hong and Materials and Methods Received for publication 19 Oct. 2002. Accepted for publication 18 Feb. 2003. We thank Kate Brilliant and Sarah Vannozzi for their technical assistance, and Joseph PLANT MATERIAL. Germplasm used in this evaluation consisted DeVerna, for helpful suggestions and critically reviewing the manuscript. of 252 clonal accessions collected between 1988 and 1993 from 1Professor; corresponding author. 2Assistant research director. Current address: USDA–ARS–Fruit Lab, Chatsworth, cultivated beds in Massachusetts, New Jersey, Oregon, Wash- NJ 08019. ington, and Wisconsin, which were established in 2.3-m2 fi eld 3Principal scientist. plots. Germplasm plots were located in one of two 0.25-ha beds 4Senior scientist. (Beds 1 and 4) at the Rutgers (N.J.) Univ. P.E. Marucci Center. J. AMER. SOC. HORT. SCI. 128(5):691–697. 2003. 691 9121-Genet 691 7/10/03, 10:59:04 AM Bed 1 has a 25-cm-deep washed sand base, while Bed 4 consists acid as a standard. The HPLC was a binary pump system (Hewlett of berryland soil. Packard 1100) with a variable diode-array detector. UV-visible FRUIT SAMPLES, TACY AND HPLC ANALYSIS. Fruit samples from spectra were recorded at 280, 320, and 520 nm. The column was 252 plots were harvested from 930-cm2 areas (minimally 30 cm from a Prodigy ODS-3, 5 µm, 250 × 4.6 mm, protected with a Prodigy plot edge) from each plot on 5 Sept. and 5 Oct. 1998. In 1999, 78 guard ODS-3, 5 µm, 30 × 4.6 mm. Separations were carried out accessions were selected (accessions were selected to represent the using the following conditions: 1 mL·min–1 fl ow rate, 25-µL injec- full range of variation based on 1998 principal component analysis) tion volume; column compartment temperature 30 °C; solvent A, for fruit samples on 20 Sept. and 11 Oct. The data obtained for each 2% (v/v) acetic acid in water; solvent B, 80% (v/v) acetonitrile and fruit sample harvested were total weight, berry count, mean fruit 20% solvent A; elution with linear gradients from 0% B (balance weight, damaged fruit weight and number and chemical analyses 100% A) for 3 min, from 0% B to 4% B in 3 min, from 4% B to are described below. 10% B in 9 min, from 10% B to 15% B in 15 min, from 15% B to For both years, fruit samples (50 g) were frozen at –20 °C. The 23% B in 20 min, from 23%B to 25% in 10 min, from 25% B to samples were analyzed randomly across plots (accessions) and 30% B in 6 min, from 30% B to 50% B in 14 min, from 50% B to harvest dates. The 50 g frozen fruit sample along with 60 mL of 80% B in 3 min, and then back to 0% B in 2 min, followed by a hot (50 °C) tap water were placed in a blender and macerated for 3 20 min post time. Individual anthocyanins were quantifi ed using min. From the resultant slurry, the total anthocyanins (TAcy) were a standard of cyanidin-3-galactoside at 520 nm. TAcy-HPLC was determined using a standard spectrophotometric assay adapted from calculated as the sum of areas representing the peaks at retention Sapers et al. (1983b). Concurrently, the sample was also prepared time (RT) for the six anthocyanins. for analysis of total phenolics, individual anthocyanins and total STATISTICAL ANALYSIS. Statistical analysis employed SAS 8.0 anthocyanins by HPLC (TAcy-HPLC). The slurry was centrifuged for Microsoft Windows, SAS Institute, Inc., Cary, N.C. Procedures (40,790 ×g, 10 min, 25 °C), the supernatant fi ltered (0.45 µm), the used were PROC CORR for Pearsonʼs correlation coeffi cients, fi ltrate sealed in a 2 mL amber glass injection vial, and frozen until PROC PRIN for principal component analysis, and PROC GLM analysis. The HPLC method used was adapted from Lamuela- for analysis of variance with Type III sums-of-squares for testing Raventos and Waterhouse (1994), substituting catechin for gallic bed, harvest date and variety effects. Table 1. Eigenvalue percentages for principal components (PC) 1–5 vectors derived from the absolute levels of six cranberry anthocyanins in the germplasm collection. 1998 1999 5 Sept. 5 Oct. Combined 20 Sept. 11 Oct. Combined N = 252 N = 245 N = 497 N = 78 N = 78 N = 156 PC 1 92.0 89.2 91.8 94.7 86.3 91.2 PC 2 5.2 6.1 4.7 3.1 7.5 4.6 PC 3 2.5 4.1 3.1 1.9 5.6 3.8 PC 4 0.2 0.4 0.2 0.2 0.4 0.3 PC 5 0.1 0.2 0.1 0.1 0.1 0.1 Table 2.